Polymers such as polyethylene glycol (PEG, Scheme 1), also named polyethylene oxide (PEO), are of great interest due to their wide range of applications in the fields of biology and medicine (Haag and Fratz (2006) Angewandte Chemie Int. Ed. 45, 1198-1215), and organic synthesis (Gravert and Janda (1997) Chemistry Reviews, 97, 489-509).

Scheme 1—structure of a polyethylene glycol (PEG) molecule. n=1, 2, 3 . . . to n
PEG (from here on, whenever PEG is referred to, it is meant such polymer of any molecular weight) has several properties that make it attractive in biotechnology and medical applications. It is water soluble, non-toxic, largely non-immunogenic, and, when covalently attached to other molecules, the resulting conjugate presents different physico-chemical properties, often resulting in improved pharmacological properties of drugs. Polymers such as PEG, and others such as polypropylene glycol, polybutylene oxide and block copolymers of these, can be used to produce polymer-protein and drug-polymer conjugates, as well as supramolecular drug-delivery systems (Pasut and Veronese (2009) Advanced Drug Delivery Reviews, 61. 1177-1188; Haag and Fratz (2006), Angewandte Chemie Int. Ed. 45, 1198-1215). U.S. Pat. No. 4,179,337, describes the improvement of blood circulation lifetime of proteins attached to PEG (a technology named PEGylation), while Haag and Fratz (2006), Angewandte Chemie Int. Ed. 45, 1198-1215, review several polymer-protein drugs that have been approved, or are currently undergoing clinical trials. PEGylation of the insoluble drug taxol yielded a water soluble conjugate that still maintained its toxicity profile (Greenwald et al. (1995), Journal of Organic Chemistry, 60, 331-336). Encapsulation of the anticancer drug doxorubicin (DOX) in a PEGylated liposome has been shown to improve DOX delivery to cancer cells and to allow for a more favourable toxicity profile (Strother and Matei (2009), Therapeutics and Clinical Risk Management. 5, 639-650).
PEGs are also used as soluble polymer supports in the synthesis of several relevant molecules, such as peptides, oligonucleotides, oligosaccharides, and small molecules. Gravert and Janda (1997), Chemistry Reviews. 97, 489-509, review the polymers, chemistries, and separation and purification methods used in liquid phase organic synthesis.
As all the above mentioned applications develop, it becomes apparent that there is a need for PEGs, and other polymers, with one or both of two important properties: (i) mono-disperse molecular weight (MW) distribution, which allows full characterisation of single molecular entities instead of mixtures, and confers well defined physical properties (Hunter and Moghimi (2002), Drug Discovery Today 7(19), 998-1001; French et al., (2009), Angewandte Chemie Int. Ed. 48, 1248-1252; Greco and Vicent (2009), Advanced Drug Delivery Reviews 61, 1203-1213), and; (ii) different functional groups at their termini (Scheme 2), allowing for efficient attachment to different molecules (proteins, peptides, oligonucleotides, small molecule drugs, liposomes, and others), as well as cross-linking between different chemical species.X-PEGn-Y
Scheme 2—structure of a heterobifunctional polyethylene glycol (PEG) molecule. n=1, 2, 3 . . . to n
Common functional groups used to replace one of the hydroxyls of PEG (X, Y, scheme 2) include aldehyde, carboxylate, trifluorosulfonate, thiol, chloride, carboxy succinimidyl esters, p-nitrophenylcarbonate, benzotriazole carbonate and others known to those skilled in the art.
Heterobifunctional PEGs are often difficult to synthesise. Oxy-anion initiated ring-opening polymerisation of ethylene oxide most commonly yields heterobifunctional PEGs with a relatively inert methoxy in one terminus and wide MW distribution. This method has been extensively used, and several disadvantages are known to those skilled in the art and have been described elsewhere (Bentley et al. (2002), U.S. Pat. No. 6,448,369B1). Reacting PEG with equimolar amounts of reagent leads to a mixture of di-, mono- and unreacted PEG, which requires tedious purification. Purification becomes increasingly more challenging as the MW of the PEG increases, due to the diminishing difference in chemical and physical properties between the di-, mono- and unreacted species. Huang (1992) Journal of Applied Polymer Science 46, 1663-1671; and Huang and Hu (1993) Journal of Applied Polymer Science 47, 1503-1511 describe the synthesis of several heterofunctionalised PEGs via a technique that uses PEG as starting material and in which, by grafting the reacted material to PVA, precipitation procedures can be used for separation and purification. Chromatographic methods with different synthetic strategies have also been applied in the synthesis of heterobifunctional PEGs (Zalipsky (1993) Bioconjugate Chemistry 4, 296-299; Phillips and Snow (1994) U.S. Pat. No. 5,298,410). U.S. Pat. No. 6,448,369B1 describes a method that avoids intermediate or final chromatographic methods by using benzyloxy-PEG-OH or arylmethyl oxy groups that can be removed under mild conditions by catalytic hydrogenolysis or acid/catalysed hydrolysis, and performing intermediate separations by ether precipitation. However, the restrictions entailed in the starting material limits the synthetic manipulations that can be undertaken and introduces PEG impurities that arise from the starting material.
The preparation of mono-disperse PEGs has been performed by the sequential conjoining of discreet short PEG oligomers, having single molecular compositions, in convergent routes to ever longer chains (Ahmed and Tanaka, (2004) J. Org. Chem., vol. 71, pages 9884-9886; French et al., (2009) Angewandte Chemie Int. Ed. 48, 1248-1252). This process shares some problems with the preparation of heterobifunctional PEGs, particularly that it becomes increasingly difficult to separate products from reactants as the molecular weight increases. Furthermore, the need to minimise purification difficulties by using near-stoichiometric combinations of PEG oligomers in the key etherification reaction has meant that reactions could not be forced to completion. Additionally, side-reactions of the starting materials, such as basic depolymerisation of ethylene oxide units from the PEG alkoxide nucleophile, and elimination of the PEG sulfonate electrophile to the corresponding vinyl ether (Loiseau et al., (2004), J. Org. Chem., vol. 69, pages 639-647), further diminished yield and product purity.
Membrane processes are well known in the art of separation science, and can be applied to a range of separations of species of varying molecular weights in liquid and gas phases (see for example “Membrane Technology” in Kirk Othmer Encyclopedia of Chemical Technology, 4th Edition 1993, Vol 16, pages 135-193). Nanofiltration is a membrane process utilising membranes whose pores are in the range 0.5-5 nm, and which have MW cutoffs of 200-3,000 Daltons. Nanofiltration has been widely applied to filtration of aqueous fluids, but due to a lack of suitable solvent stable membranes has not been widely applied to separation of solutes in organic solvents. Ultrafiltration membranes typically have MW cutoffs in the range 3,000 to 1,000,000 Daltons. Recently new classes of membranes have been developed which are stable in even the most difficult solvents as reported in P. Vandezande, L. E. M. Gevers and I. F. J. Vankelecom Chem. Soc. Rev., (2008), Vol 37, pages 365-405. These may be polymeric membranes or ceramic membranes, or mixed inorganic/organic membranes.
It is an object of the present invention to provide an improved process for the preparation of heterobifunctional polymers.
It is a further object of the present invention to provide an improved process for the preparation of mono-disperse polymers or polymers with narrow molecular weight distributions.